Background Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) have been a major tool in the malaria elimination efforts, but their use is complicated by false-negative results in regions where Plasmodium falciparum parasites have deletions in the pfhrp2 and pfhrp3 genes. But information about the prevalence and significance of these deletions is still limited in Bangladesh. Given the recent reports of diagnostic discordant P. falciparum infections from various locations in the malaria-endemic district of Bandarban, which has the highest malaria burden in Bangladesh, we sought to confirm pfhrp2 and pfhrp3 gene deletions in suspected samples. Methods A descriptive study was carried out in the laboratory with clinical samples of P. falciparum from Bandarban from 2023 to 2025. Twenty-two samples from symptomatic persons who were negative by routine HRP2-based RDTs, but positive by microscopy and Plasmodium species-specific polymerase chain reaction (PCR). Microscopy was used to determine parasite densities. The deletion of pfhrp2 , pfhrp3 and selected flanking loci were determined by conventional PCR and confirmed by probe-based real-time PCR. Results Microscopic examination revealed parasite densities ranging from 440 to 215,000 parasites/µL. Conventional PCR demonstrated pfhrp2 gene deletion in all isolates, with 20 (90.9%) of 22 also exhibiting pfhrp3 deletion, indicating a predominance of double-deletion parasites. Probe-based real-time PCR also confirmed pfhrp2 deletion in all isolates, while only two samples retained detectable pfhrp3 signals. Analysis of flanking regions revealed that genomic deletions extended beyond the target genes, with conserved loss patterns at the pfhrp2 locus. Conclusions This study shows significant pfhrp2 gene deletions and frequent co-deletion of pfhrp3 in P. falciparum isolates from Bandarban, with large genomic deletions in the flanking regions. These findings provide the most likely explanation for the observed false-negative HRP2-based RDT results and signal a potentially major threat to the current malaria diagnostic approaches in this high-endemic region. A targeted survey for pfhrp2/3 deletions is needed to assess the scale and scope of the threat in parallel with careful consideration for alternative interim and long-term diagnostic strategies to ensure accurate case detection and to support effective malaria elimination efforts in Bangladesh.

Background:COVID-19 antigen-based rapid diagnostic tests (Ag-RDTs) for self-testing (C19ST) have been widely implemented. However, evidence on population health and implementation outcomes remains limited. We systematically evaluated population health and implementation outcomes of C19ST to inform WHO guidelines and pandemic preparedness. Methods:We conducted a systematic review and meta-analysis (PROSPERO CRD42022299977), searching Embase, MEDLINE, Web of Science, MedRxiv, clinicaltrials.gov and the Cochrane Library from Dec 1, 2020, to Oct 1, 2025. We included cohort, case-control, cross-sectional, before-and-after, and randomised studies with symptomatic and asymptomatic participants using commercially available C19ST Ag-RDTs. Primary outcomes included C19ST population health (case detection, test positivity, number needed to test (NNT)) and implementation outcomes (uptake, adherence, result reporting). Meta-analyses used binomial-normal generalised linear mixed models; study quality assessment used the JBI Quasi-Experimental Tool. Findings:Of 19,473 records screened, 61 studies (87 datasets) with 25,288,225 participants (78% asymptomatic) were included. C19ST detected 31 (95% CI 14-65) cases per 1000 individuals, missing 14% (95% CI 1-65%) compared to molecular testing. Test positivity was 7 per 1000 tests (95% CI 3-15); false positives occurred in 0.4% (95% CI 0.2-1.0%). NNT was 75 in symptomatic and 1002 in asymptomatic individuals. Uptake, adherence, and result reporting were high, but estimates were limited by selection bias. C19ST was also reported to improve perceptions of safety, and reduce self-isolation, workplace absenteeism, and other societal disruptions, supporting the continuity of daily activities. Heterogeneity was substantial. Interpretation:C19ST improves case detection and supports pandemic control with acceptable accuracy and meaningful societal benefits. These findings support the use of antigen-based self-testing as a complementary tool for a pandemic response. Studies included further highlight the limited use of standardised frameworks for evaluating population health and implementation outcomes of novel diagnostics. Funding:Ministry of Science, Research and Arts of the State of Baden-Wuerttemberg, Germany.

BACKGROUND:Approximately 74% of malaria diagnoses worldwide rely on Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based rapid diagnostic tests (RDTs), whose accuracy can be compromised by deletions in its encoding pfhrp2 gene and its paralogous pfhrp3 genes. Estimating the burden of false-negative RDT results due to pfhrp2 deletion among individuals with malaria symptoms is critical for guiding the continued use of PfHRP2 tests. METHODS:This cross-sectional survey obtained 5394 samples from individuals presenting with symptoms of malaria at selected health facilities in ten counties, Kenya. Each participant was tested using both PfHRP2-based and P. falciparum lactate dehydrogenase (PfLDH)-based RDT. Samples that tested positive by PfLDH and negative by PfHRP2 were scored as discordant. All discordant samples, plus a random subset of the Plasmodium positive samples, were sequenced to characterize HRP2/3 gene deletion as well as polymorphisms in the Plasmodium falciparum kelch 13 (pfk13) propeller gene. RESULTS:The HRP2- and PfLDH-based RDTs showed similar positivity rates (44% vs. 45%), with 1.26% discordance. Discordance was highest in Nairobi, Trans Nzoia, and Kisumu, and minimal in Kwale and Tana River. None of the discordant samples carried pfhrp2 deletions, although 5.4% (3/56) had single pfhrp3 deletions, comparable to concordant infections. Among 598 samples that were sequenced, single pfhrp2 and pfhrp3 deletion prevalences were 4.5% and 12.4%, respectively, with no double-deletions detected. Nairobi exhibited the highest prevalence of pfhrp2-only deletions. Tana River, Garissa, Kirinyaga, and Kisii had the highest pfhrp3-only deletions. Among discordant samples, PfLDH-based RDTs identified P. ovale and P. malariae. Finally, sequencing of pfk13 in a subset of P. falciparum infections revealed 0.7% mutation prevalence, comprising A578S (n = 2) Kisumu and A675V (n = 2) each from Nairobi and Kirinyaga counties. CONCLUSIONS:Although single pfhrp2 and pfhrp3 deletions were observed across Kenya, they do not appear to be the primary cause of false-negative HRP2-based RDT results. Continued absence of parasites with double pfhrp2/pfhrp3 deletions supports the ongoing reliability of HRP2-based RDTs for P. falciparum detection. Routine molecular surveillance remains critical to monitor emerging deletion trends. The detection of the A675V mutation, a WHO-validated marker of partial artemisinin resistance in two counties, suggests limited spread of artemisinin resistance.

Since their first detection in 2010, Plasmodium falciparum malaria parasites lacking the P. falciparum histidine-rich protein 2 gene (pfhrp2) have been observed in 40 of 47 surveyed countries, as documented by the World Health Organization. These genetic deletions reduce detection by the most widely used rapid diagnostic tests, prompting three countries to switch to alternative diagnostics. However, manufacturing of alternative rapid diagnostic tests has not been scaled up and there are no World Health Organization-prequalified combination tests that use P. falciparumPlasmodium lactate dehydrogenase. The continuing spread of pfhrp2 and/or pfhrp3 (pfhrp2/3) deletions threatens malaria control, creating an emerging public health crisis. Here we use mathematical modeling informed by current pfhrp2/3 deletion prevalence and a literature review to assess the global risk of pfhrp2/3 deletions. We identify ten priority countries for surveillance and predict that the primary spread in Africa will move southward from the Horn of Africa through East Africa within 20 years. Despite variation in modeled timelines due to uncertainty in model parameters, four countries yet to switch rapid diagnostic tests are consistently classified as high risk under a range of model assumptions. This updated model offers refined predictions to guide pfhrp2/3 policy and prioritize future surveillance efforts and innovation.

BACKGROUND:The World Health Organization (WHO) recommends parasite-based diagnosis of malaria before treatment. The use of nucleic-acid amplification (NAAT) for detection of Plasmodium spp. has expanded rapidly in recent years, for epidemiological research globally and clinical care in high-resource settings. Data from NAATs are frequently used to inform policy decisions, so quality control is essential to ensure results are reliable and comparable. Therefore, robust quality control, including an external quality assessment (EQA) scheme targeting malaria NAATs, is essential. The WHO Global Malaria Programme and the UK National External Quality Assessment Service (UK NEQAS) have collaborated since 2017 to implement a global malaria NAAT EQA scheme. METHODS:Panels of specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were validated by expert referee laboratories prior to distribution. Between 37 and 51 laboratories participated in each distribution and submitted results online. Participants were scored based on their laboratory's stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers. Change in performance over time was calculated using a generalized mixed model with a logit link function. RESULTS:Participating laboratories were located in 42 countries. Sample format (DBS or LB) and parasite density were found to significantly affect performance, while referee labs performed better at identifying P. falciparum samples than non-referee labs. Performance of laboratories improved significantly over time, especially for lower density and P. falciparum samples. CONCLUSIONS:Results from the first eleven distributions indicate that the EQA scheme has facilitated improved performance of laboratories over time, highlighting the value of implementing such programmes. EQA schemes are critical to safeguarding the reliability of data and diagnoses, especially in situations where NAAT methodologies and protocols are used. In future, funders should make participation in an EQA scheme a requirement for laboratories, and countries can take initiatives to embed such schemes into their own national assessment programmes.

BACKGROUND:Accurate rapid diagnostic tests for SARS-CoV-2 infection could help manage the COVID-19 pandemic by potentially increasing access to testing and speed detection of infection, as well as informing clinical and public health management decisions to reduce transmission. Previous iterations of this review provided clear and conclusive evidence of superior test performance in those experiencing possible signs and symptoms of Covid-19. However, test performance in asymptomatic individuals and sensitivity by setting and indication for testing remains unclear. This is the fourth iteration of this review, first published in 2020. OBJECTIVES:To assess the diagnostic accuracy of rapid, point-of-care antigen tests (Ag-RDTs) for diagnosis of SARS-CoV-2 infection in asymptomatic population groups. SEARCH METHODS:We searched the COVID-19 Open Access Project living evidence database from the University of Bern (which includes daily updates from MEDLINE and Embase and preprints from medRxiv and bioRxiv) on 17 February 2022. We included independent evaluations from national reference laboratories, FIND and the Diagnostics Global Health website. We did not apply language restrictions. SELECTION CRITERIA:We included test accuracy studies of any design that evaluated commercially produced, rapid antigen tests in asymptomatic people tested because of known or suspected contact with SARS-CoV-2 infection, known SARS-CoV-2 infection or known absence of infection, or those who were being screened for infection. We included evaluations of single applications of a test (one test result reported per person). Reference standards for presence or absence of infection were any laboratory-based molecular test (primarily reverse transcription polymerase chain reaction (RT-PCR)). DATA COLLECTION AND ANALYSIS:We used standard screening procedures with three reviewers. Two reviewers independently carried out quality assessment (using the QUADAS-2 tool) and extracted study results. Other study characteristics were extracted by one review author and checked by a second. We present sensitivity and specificity with 95% confidence intervals (CIs) for each test, and pooled data using the bivariate model. We investigated heterogeneity by including indicator variables in the random-effects logistic regression models. We tabulated results by test manufacturer and compliance with manufacturer instructions for use and according to symptom status. MAIN RESULTS:We included 146 study cohorts (described in 130 study reports). The main results relate to 164 evaluations of single test applications including 144,250 unique samples (7104 with confirmed SARS-CoV-2) obtained from asymptomatic or mainly asymptomatic populations. Studies were mainly conducted in Europe (85/146, 58%), and evaluated 41 different commercial antigen assays (test kit). Only six studies compared two or more brands of test. Nearly all studies (96%) used RT-PCR alone to define presence or absence of infection. Risk of bias was high because of participant selection (13, 9%); interpretation of the index test (3, 2%); weaknesses in the reference standard for absence of infection (3, 2%); and participant flow and timing (46, 32%). Characteristics of participants (11, 8%) and index test delivery (117, 80%) differed from the way in which and in whom the test was intended to be used. Estimates of sensitivity varied considerably between studies, with consistently high specificities. Average sensitivity was 55.0% (95% CI 50.9%, 59.0%) and average specificity was 99.5% (95% CI 99.5%, 99.6%) across the 147 evaluations of Ag-RDTs reporting both sensitivity and specificity (149,251 samples, 7636 cases). Average sensitivity was higher when epidemiological exposure to SARS-CoV-2 was suspected (58.6%, 95% CI 51.4% to 65.5%; 43 evaluations; 15,516 samples, 1483 cases) compared to where COVID-19 testing was reported to be widely available to anyone on presentation for testing (53.0%, 95% CI 48.4% to 57.5%; 103 evaluations; 129,032 samples, 5660 cases); however CIs overlapped, limiting the inference that can be drawn from these data. Average specificity was similarly high for both groups (99.4% and 99.6%). Sensitivity was generally lower when used in a screening context (summary values from 40.6% to 42.1% for three of four screening settings) compared to testing asymptomatic individuals at Covid-19 test centres (56.7%) or emergency departments (54.7%). We observed a decline in summary sensitivities as measures of sample viral load decreased. Sensitivity varied between brands. When tests were used according to manufacturer instructions, average sensitivities by brand ranged from 36.3% to 78.8% in asymptomatic participants (14 assays with sufficient data for pooling). None of the assays met the WHO acceptable performance standard for sensitivity (of 80%) based on meta-analysis; however, sensitivities from individual studies (where meta-analysis was not possible) exceeded 80% for three assays. The WHO acceptable performance criterion of 97% specificity was met by all but four assays (based on individual studies or meta-analysis) when tests were used according to manufacturer instructions. At 0.5% prevalence using summary data for asymptomatic people, where testing was widely available and where epidemiological exposure to COVID-19 was suspected, resulting PPVs would be 40% and 33%, meaning that 3 in 5 or 2 in 3 positive results will be false positives, and between 1 in 2 and 2 in 5 cases will be missed. AUTHORS' CONCLUSIONS:Evidence for antigen testing in asymptomatic cohorts has increased considerably since the publication of the previous update of this review. Average sensitivities remain lower for testing of asymptomatic when compared to symptomatic individuals; however, there is an indication that sensitivities may be higher where epidemiological exposure to SARS-CoV-2 is suspected compared to testing any asymptomatic individual regardless of indication. Sensitivities were particularly low when antigen tests were used in screening settings. Assays from different manufacturers also vary in sensitivity, indicating the need for appropriate clinical validation of a particular antigen test in a given intended use setting prior to more widespread deployment. Further research is needed to evaluate the effectiveness of screening programmes at reducing transmission of infection, whether mass screening or targeted approaches, including schools, healthcare setting and traveller screening. FUNDING:This paper presents independent research supported by the NIHR Birmingham Biomedical Research Centre, University Hospitals Birmingham NHS Foundation Trust, and the University of Birmingham. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. REGISTRATION:Protocol (2020) doi: 10.1002/14651858.CD013596.

In the thirteen years since the first report of pfhrp2-deleted parasites in 2010, the World Health Organization (WHO) has found that 40 of 47 countries surveyed worldwide have reported pfhrp2/3 gene deletions. Due to a high prevalence of pfhrp2/3 deletions causing false-negative HRP2 RDTs, in the last five years, Eritrea, Djibouti and Ethiopia have switched or started switching to using alternative RDTs, that target pan-specific-pLDH or P. falciparum specific-pLDH alone of in combination with HRP2. However, manufacturing of alternative RDTs has not been brought to scale and there are no WHO prequalified combination tests that use Pf-pLDH instead of HRP2 for P. falciparum detection. For these reasons, the continued spread of pfhrp2/3 deletions represents a growing public health crisis that threatens efforts to control and eliminate P. falciparum malaria. National malaria control programmes, their implementing partners and test developers desperately seek pfhrp2/3 deletion data that can inform their immediate and future resource allocation. In response, we use a mathematical modelling approach to evaluate the global risk posed by pfhrp2/3 deletions and explore scenarios for how deletions will continue to spread in Africa. We incorporate current best estimates of the prevalence of pfhrp2/3 deletions and conduct a literature review to estimate model parameters known to impact the selection of pfhrp2/3 deletions for each malaria endemic country. We identify 20 countries worldwide to prioritise for surveillance and future deployment of alternative RDT, based on quickly selecting for pfhrp2/3 deletions once established. In scenarios designed to explore the continued spread of deletions in Africa, we identify 10 high threat countries that are most at risk of deletions both spreading to and subsequently being rapidly selected for. If HRP2-based RDTs continue to be relied on for malaria case management, we predict that the major route for pfhrp2 deletions to spread is south out from the current hotspot in the Horn of Africa, moving through East Africa over the next 20 years. We explore the variation in modelled timelines through an extensive parameter sensitivity analysis and despite wide uncertainties, we identify three countries that have not yet switched RDTs (Senegal, Zambia and Kenya) that are robustly identified as high risk for pfhrp2/3 deletions. These results provide a refined and updated prediction model for the emergence of pfhrp2/3 deletions in an effort to help guide pfhrp2/3 policy and prioritise future surveillance efforts and innovation.

Monkeypox virus (MPXV) is endemic in western and Central Africa, and in May 2022, a clade IIb lineage (B.1) caused a global outbreak outside Africa, resulting in its detection in 116 countries and territories. To understand the global phylogenetics of MPXV, we analyzed all available MPXV sequences, including 10,670 sequences from 65 countries collected between 1958 and 2024. Our analysis reveals high mobility of clade I viruses within Central Africa, sustained human-to-human transmission of clade IIb lineage A viruses within the Eastern Mediterranean region and distinct mutational signatures that can distinguish sustained human-to-human from animal-to-animal transmission. Moreover, distinct clade I sequences from Sudan suggest local MPXV circulation in areas of eastern Africa over the past four decades. Our study underscores the importance of genomic surveillance in tracking spatiotemporal dynamics of MXPV clades and the need to strengthen such surveillance, including in some parts of eastern Africa. An analysis of all available mpox virus sequences, including 10,670 sequences from 65 countries collected between 1958 and 2024, unveils the circulation pattern and spatiotemporal dynamics underlying the spread of the different viral clades.

The world has seen unprecedented gains in the global genomic surveillance capacities for pathogens with pandemic and epidemic potential within the last 4 years. To strengthen and sustain the gains made, WHO is working with countries and partners to implement the Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 2022-2032. A key technical product developed through these multi-agency collaborative efforts is a genomics costing tool (GCT), as sought by many countries. This tool was developed by five institutions - Association of Public Health Laboratories, FIND, The Global Fund to Fight AIDS, Tuberculosis and Malaria, UK Health Security Agency, and the World Health Organization. These institutions developed the GCT to support financial planning and budgeting for SARS-CoV-2 next-generation sequencing activities, including bioinformatic analysis. The tool costs infrastructure, consumables and reagents, human resources, facility and quality management. It is being used by countries to (1) obtain costs of routine sequencing and bioinformatics activities, (2) optimize available resources, and (3) build an investment case for the scale-up or establishment of sequencing and bioinformatics activities. The tool has been validated and is available in English and Russian at https://www.who.int/publications/i/item/9789240090866. This paper aims to highlight the rationale for developing the tool, describe the process of the collaborative effort in developing the tool, and describe the utility of the tool to countries.

Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.

Background Rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein-2 (PfHRP2) are exclusively deployed in Uganda, but deletion of the pfhrp2/3 target gene threatens their usefulness as malaria diagnosis and surveillance tools. Methods A cross-sectional survey was conducted at 40 sites across four regions of Uganda in Acholi, Lango, W. Nile and Karamoja from March 2021 to June 2023. Symptomatic malaria suspected patients were recruited and screened with both HRP2 and pan lactate dehydrogenase (pLDH) detecting RDTs. Dried blood spots (DBS) were collected from all patients and a random subset were used for genomic analysis to confirm parasite species and pfhrp2 and pfhrp3 gene status. Plasmodium species was determined using a conventional multiplex PCR while pfhrp2 and pfhrp3 gene deletions were determined using a real-time multiplex qPCR. Expression of the HRP2 protein antigen in a subset of samples was further assessed using a ELISA. Results Out of 2435 symptomatic patients tested for malaria, 1504 (61.8%) were positive on pLDH RDT. Overall, qPCR confirmed single pfhrp2 gene deletion in 1 out of 416 (0.2%) randomly selected samples that were confirmed of P. falciparum mono-infections. Conclusion These findings show limited threat of pfhrp2/3 gene deletions in the survey areas suggesting that HRP2 RDTs are still useful diagnostic tools for surveillance and diagnosis of P. falciparum malaria infections in symptomatic patients in this setting. Periodic genomic surveillance is warranted to monitor the frequency and trend of gene deletions and its effect on RDTs.

OBJECTIVES:The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. METHODS:Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. RESULTS:qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. CONCLUSIONS:Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

The World Health Organization framework for tracking SARS-CoV-2 variants has been updated to reflect the continued evolution of the virus; this framework could be adapted for other emerging respiratory diseases with epidemic and pandemic potential.

BACKGROUND:Eritrea was the first African country to discontinue the use of histidine rich protein 2 (HRP2)-detecting rapid diagnostic tests (RDTs) for malaria diagnosis following reports of a high prevalence of pfhrp2/3-deleted Plasmodium falciparum parasites causing false-negative results in the country. Eritrea was also the first African country to report partial artemisinin resistance due to the P falciparum kelch13 (pfk13) Arg622Ile mutation. We aimed to characterise the spatial distribution of pfk13 mutants and their interactions with pfhrp2/3 deletions in Eritrea and to assess the role of the use of HRP2-detecting RDTs and antimalarial (artesunate-amodiaquine) therapy in the spread of the two variants. METHODS:We conducted a retrospective molecular epidemiological analysis of pfk13 mutations and pfhrp2/3 deletions in existing P falciparum-infected blood samples collected as part of previous pfhrp2/3 deletion and severe malaria studies. Samples were collected in March, 2016 and between September, 2018, and January, 2020, from symptomatic patients seeking care at 15 health centres in four administration zones (Semenawi Keyih Bahri, Gash Barka, Anseba, and Debub) in Eritrea. A fragment spanning the propeller region of pfk13 was amplified from samples and sequenced using Sanger sequencing or targeted amplicon sequencing to identify genetic mutations. Deletions of pfhrp2/3 genes in samples were determined using multiplex quantitative PCR. Parasite haplotypes and genetic relatedness of parasite haplotypes were determined previously using microsatellite marker typing. The primary objective was to determine the prevalence of pfk13 mutations at health centres and administrative zones. The secondary objective was to investigate whether pfk13 mutants and pfhrp2/3 deleted parasites converge. FINDINGS:We sequenced 50 samples collected in March, 2016 from the Semenawi Keyih Bahri zone and identified no pfk13 mutations. By contrast, in 587 samples included in this study that were collected from health centres in Gash Barka, Anseba, and Debub in 2018-20, we detected five different single non-synonymous mutations: Glu605Lys, Arg622Ile, Asn657Lys, Lys658Glu, and Ser679Leu. The most prevalent mutation was pfk13 Arg622Ile, which was detected in samples collected from all nine health centres where more than five samples were available across all three administration zones, with an overall prevalence of 11·9% (70 of 587 samples; range 5·9-28·0%). We identified 22 unique pfk13 Arg622Ile mutant haplotypes among 26 samples tested, of which 13 (59·1%) were genetically related, whereas the remaining nine (40·9%) were not. The prevalence of pfk13 Arg622Ile was significantly higher in parasites with a single pfhrp3 deletion (46 [18·0%] of 255 samples) than in parasites without pfhrp2/3 deletions (ten [6·2%] of 161 samples; odds ratio 3·89 [95% CI 1·59-7·61]; p=0·0006) and with dual pfhrp2/3-deleted parasites (13 [9·0%] of 145; 2·23 [1·13-4·68]; p=0·018). INTERPRETATION:The geographical spread of the pfk13 Arg622Ile mutation might have initially resulted from the clonal expansion and spread of pfhrp2/3 deletions under the test-and-treat policy using HRP2-detecting RDTs. Subsequently, selective pressure from artemisinin combination therapy could have further facilitated the spread of both pfk13 Arg622Ile and pfhrp2/3 deletions. Continuous monitoring of trends in pfk13 and pfhrp2/3 variants is needed to inform effective malaria control and elimination strategies in Eritrea and other African countries. FUNDING:US Department of Defense Armed Forces Health Surveillance Division, Global Emerging Infections Surveillance Branch (AFHSD/GEIS), and Wellcome Trust.

Setting up a global SARS-CoV-2 surveillance system requires an understanding of how virus isolation and propagation practices, use of animal or human sera, and different neutralisation assay platforms influence assessment of SARS-CoV-2 antigenicity. In this study, with the contribution of 15 independent laboratories across all WHO regions, we carried out a controlled analysis of neutralisation assay platforms using the first WHO International Standard for antibodies to SARS-CoV-2 variants of concern (source: NIBSC). Live virus isolates (source: WHO BioHub or individual labs) or spike plasmids (individual labs) for pseudovirus production were used to perform neutralisation assays using the same serum panels. When comparing fold drops, excellent data consistency was observed across the labs using common reagents, including between pseudovirus and live virus neutralisation assays (RMSD of data from mean fold drop was 0.59). Utilising a Bayesian model, geometric mean titres and assay titre magnitudes (offsets) can describe the data efficiently. Titre magnitudes were seen to vary largely even for labs within the same assay group. We have observed that overall, live Microneutralisation assays tend to have the lowest titres, whereas Pseudovirus Neutralisation have the highest (with a mean difference of 3.2 log2 units between the two). These findings are relevant for laboratory networks, such as the WHO Coronavirus Laboratory Network (CoViNet), that seek to support a global surveillance system for evolution and antigenic characterisation of variants to support monitoring of population immunity and vaccine composition policy.

AbstractThe emergence and spread of drug- and diagnostic-resistantPlasmodium falciparumare major impediments to malaria control and elimination. We deep sequenced known drug resistance mutations and other informative loci across the genome of 609 samples collected during a study across three regions of Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused byP. falciparumcarrying the candidate artemisinin partial-resistanceK13622I mutation, which occurred less commonly in diagnostic-resistantpfhrp2/3-deleted than normal non-deleted parasites (p=0.03). Identity-by-descent analysis showed that 622I parasites were significantly more related than wild-type (p<0.001), consistent with recent expansion and spread.Pfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Parasites carrying bothpfhrp2/3deletion and 622I mutation were observed in some sites. These findings raise concern for future spread of combined drug- and diagnostic-resistant parasites and warrant close monitoring.

ABSTRACT Eritrea is the first African country to switch away from exclusive use of HRP2-based RDTs for the detection of P. falciparum due to high prevalence of pfhrp2 / 3- deleted P. falciparum parasites causing false-negative RDT results. While heavy reliance on malaria RDTs played a significant role in the rapid expansion of pfhrp2/3 -deleted parasites in Eritrea, we hypothesize that the use of antimalarial (artesunate-amodiaquine) may have also contributed to their spread. We conducted a retrospective investigation of mutations in the propeller domain of the P. falciparum kelch13 gene in samples collected in 2016 (n=50) from the Northern Red Sea Zone before the RDT switch away from HRP2-RDTs and in samples collected in 2018-2020 (n=587) from the Gash Barka, Anseba and Debub Zones after the RDT switch. No mutations were identified in the 2016 samples. However, in 2018-2019 samples, we detected five different single non-synonymous mutations. The most prevalent mutation was pfk13 R622I, which was detected in samples collected from all health centres, with an overall prevalence of 11.9% (ranging from 5.9% to 28%). Parasites carrying the R622I mutation have diverse microsatellite marker haplotypes, indicating that they had evolved multiple times from different genetic backgrounds. The prevalence pfk13 R622I was significantly higher in single pfhrp3 -deleted parasites (18.0%) compared to parasites without pfhrp2/3 deletions (6.2%) and dual pfhrp2/3 -deleted parasites (9.0%), suggesting association between the pfk13 R622I mutation and the pfhrp2/3 deletions in Eritrea. Continuous monitoring the trends in pfhrp2/3 and pfk13 mutants is needed to inform effective malaria management strategies in Eritrea.